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Xiaoaiping combined with cisplatin can inhibit proliferation and invasion and induce cell cycle arrest and apoptosis in human ovarian cancer cell lines
Biomedicine & Pharmacotherapy 89 (2017) 1172–1177
Available online at
ScienceDirect www.sciencedirect.com
Original article
Xiaoaiping combined with cisplatin can inhibit proliferation and invasion and induce cell cycle arrest and apoptosis in human ovarian cancer cell lines Ai-Wen Zhenga , Ya-Qing Chena , Jing Fanga , Ying-Li Zhanga , Dong-Dong Jiab,* a b
Department of Gynecologic Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, PR China Department of Bone and Soft-Tissue Surgery, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 January 2017 Received in revised form 5 March 2017 Accepted 5 March 2017
Purpose: The aim of the present study was utilizing Xiaoaiping as a single agent or combined with cisplatin to study its effect on the ovarian cancer cells (HO-8910 and HO-8910PM cells) in tumor cell proliferation, cell apoptosis, cell cycle distribution and cell invasion and migration. Methods: Both HO-8910 and HO-8910PM cell lines were treated with Xiaoaiping injection, cisplatin or combination. Effects on the cell viability and apoptosis induction were estimated using the Cell counting Kit-8 assay and Annexin V-FITC/Propidium Iodide staining. The distribution of cells in different phases of the cell cycle was assayed using flow cytometry analysis. The effects of Xiaoaiping on the inhibition of cell invasion and migration were researched with Transwell cell migration. Results: Both Xiaoaiping and cisplatin significantly decreased the HO-8910 cell survival versus control arm. Combination treatment showed a higher cytotoxicity to cells in vitro than Xiaoaiping and cisplatin alone. An increase in the G0/G1 phase fraction in HO-8910 cells treated with either Xiaoaiping or cisplatin alone was evident when compared to the fraction in control arm. Compared to the effects of treatment with either agent alone, combination treatment significantly increased the fraction of cells in G0/G1 phase. The HO-8910 cell lines treated with cisplatin demonstrated a significant increase of apoptotic cell rate compared to untreated cell lines. The rate of apoptosis in combined treatment group was significantly higher than that of the single agent treatment groups. A significant reduction in the number of invading cells was observed for Xiaoaiping-treated HO-8910 cells compared with the control group. However, cisplatin did not significantly induce the migration of cells versus untreated cells. Combination of Xiaoaiping and cisplatin significantly suppressed cell invasion and migration versus single-drug treatment in HO-8910 cells. The results of HO-8910PM cells were similar with HO-8910 cells in all tests. Conclusions: In summary, our results showed that Xiaoaiping as a single agent or combined with cisplatin could induce cell cycle arrest, cause apoptosis and necrosis in ovarian cancer cells, and inhibit cell invasion and migration. The present study also laid a foundation for further investigation involving molecular mechanism. Above all, it may provide a novel therapeutic regimen for ovarian cancer. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Xiaoaiping Ovarian cancer Proliferation Apoptosis Cell cycle arrest Cell invasion
1. Introduction Ovarian cancer, representing the second most common gynaecological malignancy with over 200,000 new cases diagnosed each year worldwide, is the fourth most frequent cause of cancer-related death in women and the most lethal gynecologic malignancy with approximately 125,000 women dying from this disease yearly.
* Corresponding author at: Department of Bone and Soft-Tissue Surgery, Zhejiang Cancer Hospital, 1 Banshan East Road, Hangzhou, Zhejiang 310022, PR China. E-mail address: [email protected] (D.-D. Jia). http://dx.doi.org/10.1016/j.biopha.2017.03.012 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
Owing to lack of specific symptoms and effective screening strategies to detect this disease, and having a tendency for early peritoneal dissemination, approximately 75% of patients are found at advanced stage (International Federation of Gynecology and Obstetrics [FIGO] stage III or IV). In addition, 5-year survival rate is only 20% and a median overall survival (OS) is 15–23 months in these patients [1,2]. Consequently, optimal treatment strategy is the key to improving outcomes for the patients with ovarian cancer. Over the past three decades, cytoreductive surgery aiming to diminish the residual tumor burden followed by systemic platinum-based chemotherapy has been considered the standard
A.-W. Zheng et al. / Biomedicine & Pharmacotherapy 89 (2017) 1172–1177
treatment for advanced-stage ovarian cancer. Although 70–80% of newly diagnosed patients can achieve a complete clinical response to primary platinum–taxane chemotherapy, approximately 60–70% of them will experience a relapse of the disease process with a median progression-free survival (PFS) of 18 months and eventually die of complications related with progressive disease [3–5]. As mentioned above, this neoplasm is recognized to be one of the most chemosensitive malignancies, suggesting the malignant cell population of ovarian cancer (HO-8910 and HO-8910PM cell lines) may be the appropriate cell lines to test novel agents. Xiaoaiping, a traditional Chinese herbal medicine, are produced by processing the roots of Marsdenia tenacissima, containing flavonoids, polysaccharides, steroidal saponins, alkaloids, resins and other chemical constituents, some of which are considered to have antitumor effect [6]. cisplatin, an antineoplastic drug, can inhibit DNA replication and gene transcription, which results in blocking protein synthesis and cell proliferation [7]. In this research, we were aimed to utilize Xiaoaiping as a single agent or combined with cisplatin to study its effect on the ovarian cancer cells (HO-8910 and HO-8910PM cells) in tumor cell proliferation, cell apoptosis, cell cycle distribution and cell invasion and migration. 2. Materials and methods 2.1. Chemicals and cell culture The Xiaoaiping injection was purchased from Nanjing Sanhome Pharmaceutical Co. LTD. Cisplatin was purchased from Qilu Pharmaceutical Co. LTD. Roswell Park Memorial Institute-1640 (RPMI-1640) medium, trypsin, fetal bovine serum (FBS) and transwell chamber were purchased from Gibco (Grand Island, NY, USA). Annexin-V-PI Apoptosis Detection Kit and Cell counting kit-8 (CCK-8) were provided by Dojindo (Kumamoto, Japan). The human ovarian cancer HO-8910 and HO-8910PM cell lines were obtained from Zhejiang Cancer Research Institute, China. All the cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were incubated at 37 C in a humidified atmosphere containing with 5% CO2. 2.2. Cytotoxicity assays
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2.4. Apoptosis assay Treatment was carried out as described above. After 24-h incubation, the cells were washed with PBS, resuspended in 300 ml binding puffer solution and incubated for 10 min in darkness with 5 ml Annexin V-FITC and 5 ml PI. Combining Annexin-V labeling with a PI staining allows differentiation among viable, early apoptotic, late apoptotic and necrotic cells. The analyses were performed by flow cytometry (BD FACS Calibur, San Diego, CA). 2.5. Cell invasion and migration assays The upper surface of the Transwell membrane was coated with 50 ml of Matrigel (1 mg/ml; BD Biosciences, Bedford, MA) and allowed to dry overnight at room temperature. For invasion assay, cells were seeded onto the upper well of each chamber at 1 105cells/ml, while 500 ml/well RPMI-1640 of supplemented with 10% FBS was added to the lower chamber of the Transwell chambers. Cells were then treated with the Xiaoaiping injection (0.5 mg/ml), cisplatin (5 mg/ml) or combination (Xiaoaiping injection: 0.5 mg/ml; cisplatin: 5 mg/ml). After incubation for 24 h, non-migratory cells at the bottom of the chamber were removed with cotton stickers. Cells that penetrated the filter were fixed with methanol for 30 min and stained with giemsa, and then washed with PBS. The number of migrating cells in five separate microscopic fields was counted under a light microscope (magnification, 200). 2.6. Statistical analysis Statistics were calculated by SPSS software (version 21.0). The group data were presented as the mean standard error of the mean. Statistical significance was analyzed by Student’s t-test and expressed as P value. All P values quoted are two sided, P < 0.05 is considered statistically significant. 3. Results 3.1. Reduction of cell viability induced by Xiaoaiping treatment To examine the impact of Xiaoaiping on cell proliferation, a proliferation assay was taken using a CCK-8 kit. Fig. 1 shows the reduction of the viability of HO-8910 and HO-8910PM cells after Xiaoaiping injection or cisplatin exposure compared to the
HO-8910 and HO-8910PM cells were placed in 96-well culture plates (5 104 cells/mL) respectively and incubated for 24 h to make sure they were adhesive, and then treated with Xiaoaiping injection (1 mg/ml), cisplatin (10 mg/ml) or combination (Xiaoaiping injection: 1 mg/ml; cisplatin: 10 mg/ml) for 48 h, while phosphate buffered solution (PBS) was added as control. Culture medium was removed and 10 ml of CCK-8 solution was added to each well, after which the cells were incubated for 2 h at 37 C. Absorbance of each well was quantified at 450 nm with a microplate reader (Bio-Rad, Hercules, CA). 2.3. Cell cycle analysis The cells were seeded in 6-well plate (1 105 cells/ml) and treated with Xiaoaiping injection (1 mg/ml) and cisplatin (10 mg/ ml) either alone or in combination(Xiaoaiping injection: 1 mg/ml; cisplatin: 10 mg/ml) for 24 h. The cells were then harvested and washed with cold PBS, fixed in ice-cold 75% ethanol overnight at 4 C. The fixed cells were collected by centrifugation and resuspended in PBS and incubated with 10 mg/L Propidium iodid (PI) at 37 C for 30 min in the dark. Analyses were done by flow cytometry (BD FACS Calibur, San Diego, CA).
Fig. 1. Effect of Xiaoaiping (MT), Cisplatin (CDDP) or combination (MT + CDDP) on the proliferation of HO-8910 and HO-8910PM cells.
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Fig. 2. (A) Flow cytometry histograms of HO-8910 cell line after treatment with Xiaoaiping (MT), Cisplatin (CDDP) or combination (MT + CDDP) (B) The percentage of cells after different treatment in G0/G1 phase in flow cytometry charts of HO-8910 cell line (C) The percentage of cells after different treatment in G0/G1 phase in flow cytometry charts of HO-8910PM cell line.
untreated cells. The viability of control arm was set to 100% so that the cell survival rate of HO-8910 cells treated with Xiaoaiping and cisplatin was (57.40 1.42) % and (64.47 2.28) % respectively. Both Xiaoaiping and cisplatin significantly decreased the HO-8910 cell survival versus control arm (P < 0.01). The viable cell number in Xiaoaiping injection combined with cisplatin group was decreased to (40.70 2.30) % of the control group. Combination treatment showed a higher cytotoxicity to HO-8910 cells in vitro than Xiaoaiping and cisplatin alone (P < 0.01). Similar results were shown in cytotoxicity of Xiaoaiping and cisplatin either alone or in combination to HO-8910PM cells (Fig. 1). 3.2. The effects of Xiaoaiping on cycle distribution The distribution of cells in the different phases of the cell cycle was assayed using flow cytometry analysis. An increase in the G0/ G1 phase fraction in HO-8910 cells treated with either Xiaoaiping (78.19 2.50%; P < 0.01 vs. control) or cisplatin (76.00 2.31%; P < 0.05 vs. control) alone was evident, when compared to the fraction in control arm(66.52 0.93%; Fig. 2). Compared to the effects of treatment with either agent alone, combination treatment (84.67 1.38%) significantly increased the fraction of cells in G0/G1 phase (P < 0.05 vs. Xiaoaiping; P < 0.01 vs. cisplatin; Fig. 2). Either single drug or combination showed a similar effect on the cell cycle distribution of HO-8910PM cells (Fig. 2C).
demonstrated a significant increase of apoptotic cell rate compared to untreated cell (P < 0.01; Fig. 3). The rate of apoptosis was significantly higher in the combined treatment group (57.66 2.58%) than that of the single agent treatment groups (P < 0.01; Fig. 3). The treatment with either single drug or combination on HO-8910PM cells showed a similar apoptosisinduced activity (Fig. 3C). 3.4. Effect of Xiaoaiping on migration of HO-8910 and HO-8910PM cells To investigate the effects of Xiaoaiping on cell invasion, we determined the ability of cells treated with Xiaoaiping injection, cisplatin or combination to invade through Matrigel in Transwell chambers. As shown in Fig. 4, a significant reduction in the number of invading cells was observed for Xiaoaiping-treated (101.60 13.50) HO-8910 cells compared with the control group (151.0 20.62; P < 0.01). However, cisplatin did not significantly induce the migration of HO-8910 cells versus untreated cells (134.60 15.03; P > 0.05). Combination of Xiaoaiping and cisplatin (26.60 18.01; P < 0.01) significantly suppressed cell invasion and migration versus single-drug treatment in HO-8910 cells. Similar results were shown in HO-8910PM cells in the Transwell migration assay (Fig. 4C). 4. Discussion
3.3. Apoptosis induced by Xiaoaiping An Annexin V/PI apoptosis kit was applied to quantify the percentage of cells undergoing apoptosis. A Xiaoaiping treatment showed a (15.93 1.76)% apoptotic HO-8910 cells, which is a significant increase compared with the controls (1.22 1.10%; P < 0.01). The HO-8910 cells treated with cisplatin (40.69 2.51%)
In the last three decades, platinum compounds remain the mainstay of both front-line therapy and management of relapsed disease [8,9]. In the early 1980s, cisplatin was considered to be a potent agent in the treatment of ovarian cancer [10]. Data were confirmed by Cochrane meta-analysis, which showed that platinum combination chemotherapy significantly reduced cancer
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Fig. 3. (A) Apoptosis in HO-8910 cells induced by Xiaoaiping (MT), Cisplatin (CDDP) or combination (MT + CDDP) induce (B) Quantitative results showing of apoptotic cells for HO-8910 cell line after different treatment (C) Quantitative results showing of apoptotic cells for HO-8910PM cell line after different treatment.
Fig. 4. (A) Photographs of cells that invaded the Matrigel after treatment with Xiaoaiping (MT), Cisplatin (CDDP) or combination (MT + CDDP) (B) The numbers of migrated HO-8910 cells after different treatment (C) The numbers of migrated HO-8910PM cells after different treatment.
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related mortality compared with nonplatinum therapy with an overall hazard ratio (HR) of 0.93 (95% CI: 0.83–1.05) for survival [11]. The cisplatin–paclitaxel combination showed a significant improvement in progression-free and overall survival in a randomized Phase III trial performed by the Gynecologic Oncology Group (GOG) compared with cisplatin plus cyclophosphamide, one of the standard chemotherapy regimens at that time [12]. Two randomized trials have demonstrated that a chemotherapy regimen consisting of carboplatin plus paclitaxel was preferred, on account of comparatively less toxicity than cisplatin plus paclitaxel, in particular emesis, nephrotoxicity and neurotoxicity. However, the trials compared carboplatin–paclitaxel versus cisplatin–paclitaxel showed equivalent efficacy, which is also highlighted by a meta-analysis of 11 trials including over 2000 patients [5,13,14]. The combined regimen of carboplatin and paclitaxel quickly became a well-tolerated and widely used standard management of ovarian cancer. Moreover, it is essential to receive the same chemotherapy regimens used in advanced cases of ovarian cancer for the majority of early-stage ovarian cancer patients [15]. To better clarify the role of paclitaxel in treatment of ovarian cancer, two studies (GOG 132 and ICON 3) have been conducted. They indicate that therapy with either single agent cisplatin or carboplatin was shown to be as effective as its combined treatment including paclitaxel [16,17]. The authors even concluded that single-agent carboplatin should be the reasonable first-line chemotherapy because of its favorable toxicity profile [17]. And that is why cisplatin was elected to be combined with Xiaoaiping to manage tumor cells in this study. The standard chemotherapy regimen leads to a long-term cure in only 10–15% of patients with advanced ovarian cancer, while majority of the remainder who initially respond to chemotherapy will still go on to relapse and develop drug resistance after frontline surgery and chemotherapy [18–20]. Once relapse has occurred the efficacy of subsequent chemotherapy is limited and cure rates have changed little in last decades [18]. Several molecular targeted agents, such as epidermal growthfactor receptor (EGFR) blockers (erlotinib, gefitinib and matuzumab), Tyrosine kinase inhibitors (TKIs) (e.g. imatinib) and monoclonal antibodies against human epidermal growth factor receptor 2 (HER2), were deemed to being of no or little clinical utility [21–23]. Thus far, two types of molecular targeted agents, anti-VEGF antibody bevacizumab and PARP inhibitor Olaparib, have been demonstrated to be impactful in some randomized clinical trials and approved by regulatory agencies. Nevertheless, all alternative drug-management approaches failed to improve overall survival versus a platinum agent (cisplatin or carboplatin) plus a taxane (paclitaxel or docetaxel). On the basis of the theories of Traditional Chinese Medicine, Marsdenia tenacissima, bitter and slightly sweet in taste and cool in nature, has shown a potent anticancer activity against lung cancer, stomach cancer, and bladder cancer [6]. It is reported that Xiaoaiping can significantly potentiate efficacies of neoadjuvant chemotherapy in patients with breast cancer, which may be related with the reduction of ER-a36 expression in breast cancer tissues [24]. However, the effect of Xiaoaiping on ovarian cancer is largely unknown. In the present study, the cell viability assay showed that Xiaoaiping as a single agent had a significant anti-tumor activity on HO-8910 and HO-8910PM cells lines, and combined treatment with cisplatin had a greater degree of influence on cell survival rate. Xiaoaiping induced apoptotic cell death in both cell lines as compared to untreated cells. Combination treatment induced a high rate of apoptotic cells which is in accordance to the results in cell viability assay. Xiaoaiping likely enhances ovarian cancer
sensitivity to cisplatin by inhibiting cell proliferation and inducing apoptosis. In this study we also researched the effect of Xiaoaiping on cell cycle distribution. Treatment with either Xiaoaiping or cisplatin alone did significantly affect the cell cycle distribution, and combination treatment had a more potent effect on cell cycle phases. These treatments may induce the cells arrest in G0/G1 phase, thus retarding cell proliferation. As we all know, the main obstruction to treating ovarian cancer is tumor invasion and metastasis, which is closely associated with cell migration. Thus, we proposed to determine whether Xiaoaiping had effects on the inhibition of cell invasion and migration in HO-8910 and HO-8910PM cells. Transwell cell migration and invasion are the typical approach to estimate cell ability of migration and invasion in vitro. In this study, we applied it to evaluate the capacity of migration and invasion of HO-8910 and HO-8910PM cells. A significant reduction in the number of invading cells was observed for Xiaoaiping-transfected cells compared with the control group, while cisplatin did not result in a significant reduction. Moreover, we showed that combined treatment more effectively suppressed cell migration of ovarian cancer cells. These results implied that Xiaoaiping may prevent the metastasis of tumors. Further research is required to determine the molecular mechanism by which Xiaoaiping and cisplatin-based chemotherapy containing Xiaoaiping have effects on tumor cell proliferation, cell apoptosis, cell cycle distribution and cell invasion and migration. In summary, our results showed that Xiaoaiping as a single agent or combined with cisplatin can induce cell cycle arrest, and cause apoptosis and necrosis in ovarian cancer cells, in particular inhibit cell invasion and migration. The present study also laid a foundation for further investigation involving molecular mechanism. Above all, it may provide a novel therapeutic regimen for ovarian cancer. Acknowledgment This study was funded by Traditional Medicine Science Research Program of Zhejiang Province (2013ZB025). References [1] R. Bell, M. Petticrew, S. Luengo, T.A. Sheldon, Screening for ovarian cancer: a systematic review, Health Technol. Assess. 2 (2) (1998) 1–84 i–iv. [2] N. Colombo, M. Peiretti, G. Parma, M. Lapresa, R. Mancari, S. Carinelli, et al., Newly diagnosed and relapsed epithelial ovarian carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up, Ann. Oncol. 21 (Suppl. 5) (2010) v23–30. [3] S.C. Rubin, T.C. Randall, K.A. Armstrong, D.S. Chi, W.J. Hoskins, Ten-year followup of ovarian cancer patients after second-look laparotomy with negative findings, Obstet. Gynecol. 93 (1) (1999) 21–24. [4] T.J. Herzog, The current treatment of recurrent ovarian cancer, Curr. Oncol. Rep. 8 (6) (2006) 448–454. [5] R.F. Ozols, B.N. Bundy, B.E. Greer, J.M. Fowler, D. Clarke-Pearson, R.A. Burger, et al., Phase III trial of carboplatin and paclitaxel compared with cisplatin and paclitaxel in patients with optimally resected stage III ovarian cancer: a Gynecologic Oncology Group study, J. Clin. Oncol. 21 (17) (2003) 3194–3200. [6] Z. Huang, Y. Wang, J. Chen, R. Wang, Q. Chen, Effect of Xiaoaiping injection on advanced hepatocellular carcinoma in patients, J. Tradit. Chin. Med. 33 (1) (2013) 34–38. [7] A. Karbownik, E. Szalek, H. Urjasz, A. Gleboka, E. Mierzwa, E. Grzeskowiak, The physical and chemical stability of cisplatin (Teva) in concentrate and diluted in sodium chloride 0.9%, Wspolczesna Onkol. 16 (5) (2012) 435–439. [8] K. Aabo, M. Adams, P. Adnitt, D.S. Alberts, A. Athanazziou, V. Barley, et al., Chemotherapy in advanced ovarian cancer: four systematic meta-analyses of individual patient data from 37 randomized trials. Advanced Ovarian Cancer Trialists’ Group, Br. J. Cancer 78 (11) (1998) 1479–1487. [9] M. Markman, Optimizing primary chemotherapy in ovarian cancer, Hematol. Oncol. Clin. N. Am. 17 (4) (2003) 957–968 viii. [10] T. Taniguchi, M. Tischkowitz, N. Ameziane, S.V. Hodgson, C.G. Mathew, H. Joenje, et al., Disruption of the Fanconi anemia-BRCA pathway in cisplatinsensitive ovarian tumors, Nat. Med. 9 (5) (2003) 568–574.
A.-W. Zheng et al. / Biomedicine & Pharmacotherapy 89 (2017) 1172–1177 [11] L. Stewart, G. Advanced Ovarian Cancer Trialists, Chemotherapy for advanced ovarian cancer. Advanced Ovarian Cancer Trialists Group, Cochrane Database Syst. Rev. 2000 (2) (2016) CD001418. [12] W.P. McGuire, W.J. Hoskins, M.F. Brady, P.R. Kucera, E.E. Partridge, K.Y. Look, et al., Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer, N. Engl. J. Med. 334 (1) (1996) 1–6. [13] A. du Bois, H.J. Luck, W. Meier, H.P. Adams, V. Mobus, S. Costa, et al., A randomized clinical trial of cisplatin/paclitaxel versus carboplatin/paclitaxel as first-line treatment of ovarian cancer, J. Natl. Cancer Inst. 95 (17) (2003) 1320–1329. [14] Chemotherapy in advanced ovarian cancer: an overview of randomised clinical trials. Advanced Ovarian Cancer Trialists Group, BMJ 303 (6807) (1991) 884–893. [15] L. Elit, A. Chambers, A. Fyles, A. Covens, M. Carey, M.F. Fung, Systematic review of adjuvant care for women with Stage I ovarian carcinoma, Cancer Am. Cancer Soc. 101 (9) (2004) 1926–1935. [16] F.M. Muggia, Sequential single agents as first-line chemotherapy for ovarian cancer: a strategy derived from the results of GOG-132, Int. J. Gynecol. Cancer 13 (Suppl. 2) (2003) 156–162. [17] G. International Collaborative Ovarian Neoplasm, Paclitaxel plus carboplatin versus standard chemotherapy with either single-agent carboplatin or cyclophosphamide, doxorubicin, and cisplatin in women with ovarian cancer: the ICON3 randomised trial, Lancet 360 (9332) (2002) 505–515. [18] S.A. Cannistra, Cancer of the ovary, N. Engl. J. Med. 351 (24) (2004) 2519–2529.
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[19] A.P. Heintz, F. Odicino, P. Maisonneuve, M.A. Quinn, J.L. Benedet, W.T. Creasman, et al., Carcinoma of the ovary FIGO 26th annual report on the results of treatment in gynecological cancer, Int. J. Gynaecol. Obstet. 95 (Suppl. 1) (2006) S161–S192. [20] B.J. Monk, R.L. Coleman, Changing the paradigm in the treatment of platinumsensitive recurrent ovarian cancer: from platinum doublets to nonplatinum doublets and adding antiangiogenesis compounds, Int. J. Gynecol. Cancer 19 (Suppl. 2) (2009) S63–7. [21] M.A. Bookman, K.M. Darcy, D. Clarke-Pearson, R.A. Boothby, I.R. Horowitz, Evaluation of monoclonal humanized anti-HER2 antibody, trastuzumab, in patients with recurrent or refractory ovarian or primary peritoneal carcinoma with overexpression of HER2: a phase II trial of the Gynecologic Oncology Group, J. Clin. Oncol. 21 (2) (2003) 283–290. [22] R.J. Schilder, M.W. Sill, X. Chen, K.M. Darcy, S.L. Decesare, G. Lewandowski, et al., Phase II study of gefitinib in patients with relapsed or persistent ovarian or primary peritoneal carcinoma and evaluation of epidermal growth factor receptor mutations and immunohistochemical expression: a Gynecologic Oncology Group Study, Clin. Cancer Res. 11 (15) (2005) 5539–5548. [23] R.L. Coleman, R.R. Broaddus, D.C. Bodurka, J.K. Wolf, T.W. Burke, J.J. Kavanagh, et al., Phase II trial of imatinib mesylate in patients with recurrent platinumand taxane-resistant epithelial ovarian and primary peritoneal cancers, Gynecol. Oncol. 101 (1) (2006) 126–131. [24] L.W. Ruan, Y.C. Deng, Study on effect of Xiaoaiping in enhancing efficacy of neoadjuvant chemotherapy for breast cancer and its mechanism, Zhongguo Zhong Yao Za Zhi 40 (4) (2015) 749–752.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Xiaoaiping combined with cisplatin can inhibit proliferation and invasion and induce cell cycle arrest and apoptosis in human ovarian cancer cell lines Ai-Wen Zhenga , Ya-Qing Chena , Jing Fanga , Ying-Li Zhanga , Dong-Dong Jiab,* a b
Department of Gynecologic Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, PR China Department of Bone and Soft-Tissue Surgery, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 January 2017 Received in revised form 5 March 2017 Accepted 5 March 2017
Purpose: The aim of the present study was utilizing Xiaoaiping as a single agent or combined with cisplatin to study its effect on the ovarian cancer cells (HO-8910 and HO-8910PM cells) in tumor cell proliferation, cell apoptosis, cell cycle distribution and cell invasion and migration. Methods: Both HO-8910 and HO-8910PM cell lines were treated with Xiaoaiping injection, cisplatin or combination. Effects on the cell viability and apoptosis induction were estimated using the Cell counting Kit-8 assay and Annexin V-FITC/Propidium Iodide staining. The distribution of cells in different phases of the cell cycle was assayed using flow cytometry analysis. The effects of Xiaoaiping on the inhibition of cell invasion and migration were researched with Transwell cell migration. Results: Both Xiaoaiping and cisplatin significantly decreased the HO-8910 cell survival versus control arm. Combination treatment showed a higher cytotoxicity to cells in vitro than Xiaoaiping and cisplatin alone. An increase in the G0/G1 phase fraction in HO-8910 cells treated with either Xiaoaiping or cisplatin alone was evident when compared to the fraction in control arm. Compared to the effects of treatment with either agent alone, combination treatment significantly increased the fraction of cells in G0/G1 phase. The HO-8910 cell lines treated with cisplatin demonstrated a significant increase of apoptotic cell rate compared to untreated cell lines. The rate of apoptosis in combined treatment group was significantly higher than that of the single agent treatment groups. A significant reduction in the number of invading cells was observed for Xiaoaiping-treated HO-8910 cells compared with the control group. However, cisplatin did not significantly induce the migration of cells versus untreated cells. Combination of Xiaoaiping and cisplatin significantly suppressed cell invasion and migration versus single-drug treatment in HO-8910 cells. The results of HO-8910PM cells were similar with HO-8910 cells in all tests. Conclusions: In summary, our results showed that Xiaoaiping as a single agent or combined with cisplatin could induce cell cycle arrest, cause apoptosis and necrosis in ovarian cancer cells, and inhibit cell invasion and migration. The present study also laid a foundation for further investigation involving molecular mechanism. Above all, it may provide a novel therapeutic regimen for ovarian cancer. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Xiaoaiping Ovarian cancer Proliferation Apoptosis Cell cycle arrest Cell invasion
1. Introduction Ovarian cancer, representing the second most common gynaecological malignancy with over 200,000 new cases diagnosed each year worldwide, is the fourth most frequent cause of cancer-related death in women and the most lethal gynecologic malignancy with approximately 125,000 women dying from this disease yearly.
* Corresponding author at: Department of Bone and Soft-Tissue Surgery, Zhejiang Cancer Hospital, 1 Banshan East Road, Hangzhou, Zhejiang 310022, PR China. E-mail address: [email protected] (D.-D. Jia). http://dx.doi.org/10.1016/j.biopha.2017.03.012 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
Owing to lack of specific symptoms and effective screening strategies to detect this disease, and having a tendency for early peritoneal dissemination, approximately 75% of patients are found at advanced stage (International Federation of Gynecology and Obstetrics [FIGO] stage III or IV). In addition, 5-year survival rate is only 20% and a median overall survival (OS) is 15–23 months in these patients [1,2]. Consequently, optimal treatment strategy is the key to improving outcomes for the patients with ovarian cancer. Over the past three decades, cytoreductive surgery aiming to diminish the residual tumor burden followed by systemic platinum-based chemotherapy has been considered the standard
A.-W. Zheng et al. / Biomedicine & Pharmacotherapy 89 (2017) 1172–1177
treatment for advanced-stage ovarian cancer. Although 70–80% of newly diagnosed patients can achieve a complete clinical response to primary platinum–taxane chemotherapy, approximately 60–70% of them will experience a relapse of the disease process with a median progression-free survival (PFS) of 18 months and eventually die of complications related with progressive disease [3–5]. As mentioned above, this neoplasm is recognized to be one of the most chemosensitive malignancies, suggesting the malignant cell population of ovarian cancer (HO-8910 and HO-8910PM cell lines) may be the appropriate cell lines to test novel agents. Xiaoaiping, a traditional Chinese herbal medicine, are produced by processing the roots of Marsdenia tenacissima, containing flavonoids, polysaccharides, steroidal saponins, alkaloids, resins and other chemical constituents, some of which are considered to have antitumor effect [6]. cisplatin, an antineoplastic drug, can inhibit DNA replication and gene transcription, which results in blocking protein synthesis and cell proliferation [7]. In this research, we were aimed to utilize Xiaoaiping as a single agent or combined with cisplatin to study its effect on the ovarian cancer cells (HO-8910 and HO-8910PM cells) in tumor cell proliferation, cell apoptosis, cell cycle distribution and cell invasion and migration. 2. Materials and methods 2.1. Chemicals and cell culture The Xiaoaiping injection was purchased from Nanjing Sanhome Pharmaceutical Co. LTD. Cisplatin was purchased from Qilu Pharmaceutical Co. LTD. Roswell Park Memorial Institute-1640 (RPMI-1640) medium, trypsin, fetal bovine serum (FBS) and transwell chamber were purchased from Gibco (Grand Island, NY, USA). Annexin-V-PI Apoptosis Detection Kit and Cell counting kit-8 (CCK-8) were provided by Dojindo (Kumamoto, Japan). The human ovarian cancer HO-8910 and HO-8910PM cell lines were obtained from Zhejiang Cancer Research Institute, China. All the cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were incubated at 37 C in a humidified atmosphere containing with 5% CO2. 2.2. Cytotoxicity assays
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2.4. Apoptosis assay Treatment was carried out as described above. After 24-h incubation, the cells were washed with PBS, resuspended in 300 ml binding puffer solution and incubated for 10 min in darkness with 5 ml Annexin V-FITC and 5 ml PI. Combining Annexin-V labeling with a PI staining allows differentiation among viable, early apoptotic, late apoptotic and necrotic cells. The analyses were performed by flow cytometry (BD FACS Calibur, San Diego, CA). 2.5. Cell invasion and migration assays The upper surface of the Transwell membrane was coated with 50 ml of Matrigel (1 mg/ml; BD Biosciences, Bedford, MA) and allowed to dry overnight at room temperature. For invasion assay, cells were seeded onto the upper well of each chamber at 1 105cells/ml, while 500 ml/well RPMI-1640 of supplemented with 10% FBS was added to the lower chamber of the Transwell chambers. Cells were then treated with the Xiaoaiping injection (0.5 mg/ml), cisplatin (5 mg/ml) or combination (Xiaoaiping injection: 0.5 mg/ml; cisplatin: 5 mg/ml). After incubation for 24 h, non-migratory cells at the bottom of the chamber were removed with cotton stickers. Cells that penetrated the filter were fixed with methanol for 30 min and stained with giemsa, and then washed with PBS. The number of migrating cells in five separate microscopic fields was counted under a light microscope (magnification, 200). 2.6. Statistical analysis Statistics were calculated by SPSS software (version 21.0). The group data were presented as the mean standard error of the mean. Statistical significance was analyzed by Student’s t-test and expressed as P value. All P values quoted are two sided, P < 0.05 is considered statistically significant. 3. Results 3.1. Reduction of cell viability induced by Xiaoaiping treatment To examine the impact of Xiaoaiping on cell proliferation, a proliferation assay was taken using a CCK-8 kit. Fig. 1 shows the reduction of the viability of HO-8910 and HO-8910PM cells after Xiaoaiping injection or cisplatin exposure compared to the
HO-8910 and HO-8910PM cells were placed in 96-well culture plates (5 104 cells/mL) respectively and incubated for 24 h to make sure they were adhesive, and then treated with Xiaoaiping injection (1 mg/ml), cisplatin (10 mg/ml) or combination (Xiaoaiping injection: 1 mg/ml; cisplatin: 10 mg/ml) for 48 h, while phosphate buffered solution (PBS) was added as control. Culture medium was removed and 10 ml of CCK-8 solution was added to each well, after which the cells were incubated for 2 h at 37 C. Absorbance of each well was quantified at 450 nm with a microplate reader (Bio-Rad, Hercules, CA). 2.3. Cell cycle analysis The cells were seeded in 6-well plate (1 105 cells/ml) and treated with Xiaoaiping injection (1 mg/ml) and cisplatin (10 mg/ ml) either alone or in combination(Xiaoaiping injection: 1 mg/ml; cisplatin: 10 mg/ml) for 24 h. The cells were then harvested and washed with cold PBS, fixed in ice-cold 75% ethanol overnight at 4 C. The fixed cells were collected by centrifugation and resuspended in PBS and incubated with 10 mg/L Propidium iodid (PI) at 37 C for 30 min in the dark. Analyses were done by flow cytometry (BD FACS Calibur, San Diego, CA).
Fig. 1. Effect of Xiaoaiping (MT), Cisplatin (CDDP) or combination (MT + CDDP) on the proliferation of HO-8910 and HO-8910PM cells.
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Fig. 2. (A) Flow cytometry histograms of HO-8910 cell line after treatment with Xiaoaiping (MT), Cisplatin (CDDP) or combination (MT + CDDP) (B) The percentage of cells after different treatment in G0/G1 phase in flow cytometry charts of HO-8910 cell line (C) The percentage of cells after different treatment in G0/G1 phase in flow cytometry charts of HO-8910PM cell line.
untreated cells. The viability of control arm was set to 100% so that the cell survival rate of HO-8910 cells treated with Xiaoaiping and cisplatin was (57.40 1.42) % and (64.47 2.28) % respectively. Both Xiaoaiping and cisplatin significantly decreased the HO-8910 cell survival versus control arm (P < 0.01). The viable cell number in Xiaoaiping injection combined with cisplatin group was decreased to (40.70 2.30) % of the control group. Combination treatment showed a higher cytotoxicity to HO-8910 cells in vitro than Xiaoaiping and cisplatin alone (P < 0.01). Similar results were shown in cytotoxicity of Xiaoaiping and cisplatin either alone or in combination to HO-8910PM cells (Fig. 1). 3.2. The effects of Xiaoaiping on cycle distribution The distribution of cells in the different phases of the cell cycle was assayed using flow cytometry analysis. An increase in the G0/ G1 phase fraction in HO-8910 cells treated with either Xiaoaiping (78.19 2.50%; P < 0.01 vs. control) or cisplatin (76.00 2.31%; P < 0.05 vs. control) alone was evident, when compared to the fraction in control arm(66.52 0.93%; Fig. 2). Compared to the effects of treatment with either agent alone, combination treatment (84.67 1.38%) significantly increased the fraction of cells in G0/G1 phase (P < 0.05 vs. Xiaoaiping; P < 0.01 vs. cisplatin; Fig. 2). Either single drug or combination showed a similar effect on the cell cycle distribution of HO-8910PM cells (Fig. 2C).
demonstrated a significant increase of apoptotic cell rate compared to untreated cell (P < 0.01; Fig. 3). The rate of apoptosis was significantly higher in the combined treatment group (57.66 2.58%) than that of the single agent treatment groups (P < 0.01; Fig. 3). The treatment with either single drug or combination on HO-8910PM cells showed a similar apoptosisinduced activity (Fig. 3C). 3.4. Effect of Xiaoaiping on migration of HO-8910 and HO-8910PM cells To investigate the effects of Xiaoaiping on cell invasion, we determined the ability of cells treated with Xiaoaiping injection, cisplatin or combination to invade through Matrigel in Transwell chambers. As shown in Fig. 4, a significant reduction in the number of invading cells was observed for Xiaoaiping-treated (101.60 13.50) HO-8910 cells compared with the control group (151.0 20.62; P < 0.01). However, cisplatin did not significantly induce the migration of HO-8910 cells versus untreated cells (134.60 15.03; P > 0.05). Combination of Xiaoaiping and cisplatin (26.60 18.01; P < 0.01) significantly suppressed cell invasion and migration versus single-drug treatment in HO-8910 cells. Similar results were shown in HO-8910PM cells in the Transwell migration assay (Fig. 4C). 4. Discussion
3.3. Apoptosis induced by Xiaoaiping An Annexin V/PI apoptosis kit was applied to quantify the percentage of cells undergoing apoptosis. A Xiaoaiping treatment showed a (15.93 1.76)% apoptotic HO-8910 cells, which is a significant increase compared with the controls (1.22 1.10%; P < 0.01). The HO-8910 cells treated with cisplatin (40.69 2.51%)
In the last three decades, platinum compounds remain the mainstay of both front-line therapy and management of relapsed disease [8,9]. In the early 1980s, cisplatin was considered to be a potent agent in the treatment of ovarian cancer [10]. Data were confirmed by Cochrane meta-analysis, which showed that platinum combination chemotherapy significantly reduced cancer
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Fig. 3. (A) Apoptosis in HO-8910 cells induced by Xiaoaiping (MT), Cisplatin (CDDP) or combination (MT + CDDP) induce (B) Quantitative results showing of apoptotic cells for HO-8910 cell line after different treatment (C) Quantitative results showing of apoptotic cells for HO-8910PM cell line after different treatment.
Fig. 4. (A) Photographs of cells that invaded the Matrigel after treatment with Xiaoaiping (MT), Cisplatin (CDDP) or combination (MT + CDDP) (B) The numbers of migrated HO-8910 cells after different treatment (C) The numbers of migrated HO-8910PM cells after different treatment.
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related mortality compared with nonplatinum therapy with an overall hazard ratio (HR) of 0.93 (95% CI: 0.83–1.05) for survival [11]. The cisplatin–paclitaxel combination showed a significant improvement in progression-free and overall survival in a randomized Phase III trial performed by the Gynecologic Oncology Group (GOG) compared with cisplatin plus cyclophosphamide, one of the standard chemotherapy regimens at that time [12]. Two randomized trials have demonstrated that a chemotherapy regimen consisting of carboplatin plus paclitaxel was preferred, on account of comparatively less toxicity than cisplatin plus paclitaxel, in particular emesis, nephrotoxicity and neurotoxicity. However, the trials compared carboplatin–paclitaxel versus cisplatin–paclitaxel showed equivalent efficacy, which is also highlighted by a meta-analysis of 11 trials including over 2000 patients [5,13,14]. The combined regimen of carboplatin and paclitaxel quickly became a well-tolerated and widely used standard management of ovarian cancer. Moreover, it is essential to receive the same chemotherapy regimens used in advanced cases of ovarian cancer for the majority of early-stage ovarian cancer patients [15]. To better clarify the role of paclitaxel in treatment of ovarian cancer, two studies (GOG 132 and ICON 3) have been conducted. They indicate that therapy with either single agent cisplatin or carboplatin was shown to be as effective as its combined treatment including paclitaxel [16,17]. The authors even concluded that single-agent carboplatin should be the reasonable first-line chemotherapy because of its favorable toxicity profile [17]. And that is why cisplatin was elected to be combined with Xiaoaiping to manage tumor cells in this study. The standard chemotherapy regimen leads to a long-term cure in only 10–15% of patients with advanced ovarian cancer, while majority of the remainder who initially respond to chemotherapy will still go on to relapse and develop drug resistance after frontline surgery and chemotherapy [18–20]. Once relapse has occurred the efficacy of subsequent chemotherapy is limited and cure rates have changed little in last decades [18]. Several molecular targeted agents, such as epidermal growthfactor receptor (EGFR) blockers (erlotinib, gefitinib and matuzumab), Tyrosine kinase inhibitors (TKIs) (e.g. imatinib) and monoclonal antibodies against human epidermal growth factor receptor 2 (HER2), were deemed to being of no or little clinical utility [21–23]. Thus far, two types of molecular targeted agents, anti-VEGF antibody bevacizumab and PARP inhibitor Olaparib, have been demonstrated to be impactful in some randomized clinical trials and approved by regulatory agencies. Nevertheless, all alternative drug-management approaches failed to improve overall survival versus a platinum agent (cisplatin or carboplatin) plus a taxane (paclitaxel or docetaxel). On the basis of the theories of Traditional Chinese Medicine, Marsdenia tenacissima, bitter and slightly sweet in taste and cool in nature, has shown a potent anticancer activity against lung cancer, stomach cancer, and bladder cancer [6]. It is reported that Xiaoaiping can significantly potentiate efficacies of neoadjuvant chemotherapy in patients with breast cancer, which may be related with the reduction of ER-a36 expression in breast cancer tissues [24]. However, the effect of Xiaoaiping on ovarian cancer is largely unknown. In the present study, the cell viability assay showed that Xiaoaiping as a single agent had a significant anti-tumor activity on HO-8910 and HO-8910PM cells lines, and combined treatment with cisplatin had a greater degree of influence on cell survival rate. Xiaoaiping induced apoptotic cell death in both cell lines as compared to untreated cells. Combination treatment induced a high rate of apoptotic cells which is in accordance to the results in cell viability assay. Xiaoaiping likely enhances ovarian cancer
sensitivity to cisplatin by inhibiting cell proliferation and inducing apoptosis. In this study we also researched the effect of Xiaoaiping on cell cycle distribution. Treatment with either Xiaoaiping or cisplatin alone did significantly affect the cell cycle distribution, and combination treatment had a more potent effect on cell cycle phases. These treatments may induce the cells arrest in G0/G1 phase, thus retarding cell proliferation. As we all know, the main obstruction to treating ovarian cancer is tumor invasion and metastasis, which is closely associated with cell migration. Thus, we proposed to determine whether Xiaoaiping had effects on the inhibition of cell invasion and migration in HO-8910 and HO-8910PM cells. Transwell cell migration and invasion are the typical approach to estimate cell ability of migration and invasion in vitro. In this study, we applied it to evaluate the capacity of migration and invasion of HO-8910 and HO-8910PM cells. A significant reduction in the number of invading cells was observed for Xiaoaiping-transfected cells compared with the control group, while cisplatin did not result in a significant reduction. Moreover, we showed that combined treatment more effectively suppressed cell migration of ovarian cancer cells. These results implied that Xiaoaiping may prevent the metastasis of tumors. Further research is required to determine the molecular mechanism by which Xiaoaiping and cisplatin-based chemotherapy containing Xiaoaiping have effects on tumor cell proliferation, cell apoptosis, cell cycle distribution and cell invasion and migration. In summary, our results showed that Xiaoaiping as a single agent or combined with cisplatin can induce cell cycle arrest, and cause apoptosis and necrosis in ovarian cancer cells, in particular inhibit cell invasion and migration. The present study also laid a foundation for further investigation involving molecular mechanism. Above all, it may provide a novel therapeutic regimen for ovarian cancer. Acknowledgment This study was funded by Traditional Medicine Science Research Program of Zhejiang Province (2013ZB025). References [1] R. Bell, M. Petticrew, S. Luengo, T.A. Sheldon, Screening for ovarian cancer: a systematic review, Health Technol. Assess. 2 (2) (1998) 1–84 i–iv. [2] N. Colombo, M. Peiretti, G. Parma, M. Lapresa, R. Mancari, S. Carinelli, et al., Newly diagnosed and relapsed epithelial ovarian carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up, Ann. Oncol. 21 (Suppl. 5) (2010) v23–30. [3] S.C. Rubin, T.C. Randall, K.A. Armstrong, D.S. Chi, W.J. Hoskins, Ten-year followup of ovarian cancer patients after second-look laparotomy with negative findings, Obstet. Gynecol. 93 (1) (1999) 21–24. [4] T.J. Herzog, The current treatment of recurrent ovarian cancer, Curr. Oncol. Rep. 8 (6) (2006) 448–454. [5] R.F. Ozols, B.N. Bundy, B.E. Greer, J.M. Fowler, D. Clarke-Pearson, R.A. Burger, et al., Phase III trial of carboplatin and paclitaxel compared with cisplatin and paclitaxel in patients with optimally resected stage III ovarian cancer: a Gynecologic Oncology Group study, J. Clin. Oncol. 21 (17) (2003) 3194–3200. [6] Z. Huang, Y. Wang, J. Chen, R. Wang, Q. Chen, Effect of Xiaoaiping injection on advanced hepatocellular carcinoma in patients, J. Tradit. Chin. Med. 33 (1) (2013) 34–38. [7] A. Karbownik, E. Szalek, H. Urjasz, A. Gleboka, E. Mierzwa, E. Grzeskowiak, The physical and chemical stability of cisplatin (Teva) in concentrate and diluted in sodium chloride 0.9%, Wspolczesna Onkol. 16 (5) (2012) 435–439. [8] K. Aabo, M. Adams, P. Adnitt, D.S. Alberts, A. Athanazziou, V. Barley, et al., Chemotherapy in advanced ovarian cancer: four systematic meta-analyses of individual patient data from 37 randomized trials. Advanced Ovarian Cancer Trialists’ Group, Br. J. Cancer 78 (11) (1998) 1479–1487. [9] M. Markman, Optimizing primary chemotherapy in ovarian cancer, Hematol. Oncol. Clin. N. Am. 17 (4) (2003) 957–968 viii. [10] T. Taniguchi, M. Tischkowitz, N. Ameziane, S.V. Hodgson, C.G. Mathew, H. Joenje, et al., Disruption of the Fanconi anemia-BRCA pathway in cisplatinsensitive ovarian tumors, Nat. Med. 9 (5) (2003) 568–574.
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